Evaluation of PTPN22 polymorphisms and Vogt-Koyanagi-Harada disease in Japanese patients.

PURPOSE
Vogt-Koyanagi-Harada (VKH) disease is an autoimmune disorder against melanocytes. Polymorphisms of the protein tyrosine phosphatase non-receptor 22 gene (PTPN22) have recently been reported to be associated with susceptibility to several autoimmune diseases. In this study, genetic susceptibility to VKH disease was investigated by screening for single nucleotide polymorphisms (SNPs) of PTPN22.


METHODS
A total of 167 Japanese patients with VKH disease and 188 healthy Japanese controls were genotyped by direct sequencing methods for six SNPs (rs3811021, rs1217413, rs1237682, rs3761935, rs3789608, and rs2243471) of PTPN22 including the uncoding exons.


RESULTS
The six SNPs in PTPN22 showed no significant association with susceptibility to VKH disease or its ocular, neurologic, or dermatological manifestation.


CONCLUSIONS
Further studies are needed to clarify the genetic mechanisms underlying VKH disease.

Corporation, Cleveland, OH), the PCR products were sequenced with Big Dye Terminator v3.1 (Applied Biosystems, Foster City, CA) using either sense or antisense primers ( Table 1). The BigDye XTerminator Purification Kit (Applied Biosystems) was used to purify the DNA from sequencing reactions. The sequencing reactions were analyzed using an ABI3130 sequencer (Applied Biosystems).
Statistical analysis: For statistical analyses, the Hardy-Weinberg equilibrium was tested for each SNP among the control subjects. Genotype frequency differences between the case and control genotypes were assessed by the χ 2 test. The calculation of linkage disequilibrium (LD) and pair-wise LD (D' value) between SNPs of the PTPN22 region and the haplotypes was performed with Haploview software, version 3.32. The maximum likelihood estimates of haplotype frequencies were estimated by pairs of unphased genotypes using the expectation-maximization (EM) algorithms in the R package 'haplo.stats' [20].

RESULTS
Allele frequencies for the six SNPs covering the gene were in Hardy-Weinberg equilibrium in both the patients and controls. The allelic frequency of each SNP in both groups was nearly equal, and no association was detected when compared independently (odds ratio, OR 1.14-1.35; Table 2). Stratifying the patients by the presence of diffuse choroiditis, sunset glow fundus, nummular chorioretinal depigmented spots, neurologic auditory involvement, meningismus, tinnitus, cerebrospinal fluid pleocytosis, or integumentary findings also revealed no evidence of association in VKH disease (data not shown). We calculated pairwise D' values for all SNP pairs in PTPN22 ( Figure 2). The pairwise D' values in the gene were nearly 1 among almost all SNP pairs, indicating the SNPs were highly associated with each other and the entire PTPN22 was contained within a single LD block. Haplotype analysis predicted and revealed that PTPN22 was not associated with VKH disease in this Japanese cohort (data not shown).

DISCUSSION
In the present study, we analyzed polymorphisms of the new candidate gene, PTPN22, in Japanese patients with VKH   disease. The gene encodes an important negative regulator of T cell activation [9]. An SNP of PTPN22, R620W (rs2476601) was reported to be associated with several autoimmune diseases such as RA, SLE, and IDDM [11,12,14,15]. However, this SNP, which disrupts an interaction between Lyp and the protein tyrosine kinase, Csk, does not exist as a polymorphism in the Japanese population [9,10,12]. Therefore, in this study, we examined six other SNPs to evaluate the susceptibility locus of PTPN22. HLA-DRB1 is a common genetic factor in autoimmune diseases (RA and IDDM). Therefore, there may be other common genetic factors in VKH disease [21]. VKH disease is considered to be an autoimmune disease against melanocytes [2][3][4][5]. In early studies, activated T lymphocytes were elevated and attacked melanocytes of ocular choroidal tissue in patients in the active phase of VKH disease [22]. Antigen-specific T-cell assay revealed that peptide fragments of the tyrosinase family proteins (tyrosinase, tyrosinase related protein 1 and 2) proliferated in T lymphocytes collected from VKH patients [4,5]. These proteins are found in human melanocytes. These antigenspecific T cell responses were detected in cells collected from HLA-DRB1*04 positive VKH patients only but not from HLA-DRB1*04 negative patients or HLA-DRB1*04 positive healthy people [7,23]. In the Japanese population, 40% of healthy people have HLA-DRB1*04 [7]. However, people having VKH disease represent only 0.01% of the Japanese population [1,7,24,25]. In addition, some patients with VKH disease are HLA-DRB1*04 negative [7]. Thus, it is believed HLA-DRB1*04 is a major susceptible gene in VKH disease. However, other minor genetic factors still remain unclear. To find other susceptible genes, we studied the tyrosinase gene (TYR), tyrosinase related protein 1 gene (TYRP1), tyrosinase related protein 2 gene (TYRP2), and interferon (IFN-γ), but we could not find any association with these genes and VKH disease [7,26]. Genetic influences of VKH were also investigated in other countries, but the etiology of the disease seems to be unresolved [27][28][29].
In this study, we found no association between PTPN22 and VKH disease in the individuals studied. Our results suggest that further molecular genetic studies are needed to detect novel genetic loci and predisposing genes and to elucidate the true genetic mechanisms underlying VKH disease. The above table is the genotype and allele frequencies of the VKH patients and healthy controls. There are no differences between patients and controls.